Abstract
BackgroundMicroRNAs (miRNAs) have been shown to play a critical role in the development and progression of nasopharyngeal carcinoma (NPC). Although accumulating studies have been performed on the molecular mechanisms of NPC, the miRNA regulatory networks in cancer progression remain largely unknown. Laser capture microdissection (LCM) and deep sequencing are powerful tools that can help us to detect the integrated view of miRNA-target network.MethodsIllumina Hiseq2000 deep sequencing was used to screen differentially expressed miRNAs in laser-microdessected biopsies between 12 NPC and 8 chronic nasopharyngitis patients. The result was validated by real-time PCR on 201 NPC and 25 chronic nasopharyngitis patients. The potential candidate target genes of the miRNAs were predicted using published target prediction softwares (RNAhybrid, TargetScan, Miranda, PITA), and the overlay part was analyzed in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological process. The miRNA regulatory network analysis was performed using the Ingenuity Pathway Analysis (IPA) software.ResultsEight differentially expressed miRNAs were identified between NPC and chronic nasopharyngitis patients by deep sequencing. Further qRT-PCR assays confirmed 3 down-regulated miRNAs (miR-34c-5p, miR-375 and miR-449c-5p), 4 up-regulated miRNAs (miR-205-5p, miR-92a-3p, miR-193b-3p and miR-27a-5p). Additionally, the low level of miR-34c-5p (miR-34c) was significantly correlated with advanced TNM stage. GO and KEGG enrichment analyses showed that 914 target genes were involved in cell cycle, cytokine secretion and tumor immunology, and so on. IPA revealed that cancer was the top disease associated with those dysregulated miRNAs, and the genes regulated by miR-34c were in the center of miRNA-mRNA regulatory network, including TP53, CCND1, CDK6, MET and BCL2, and the PI3K/AKT/ mTOR signaling was regarded as a significant function pathway in this network.ConclusionOur study presents the current knowledge of miRNA regulatory network in NPC with combination of bioinformatics analysis and literature research. The hypothesis of miR-34c regulatory pathway may be beneficial in guiding further studies on the molecular mechanism of NPC tumorigenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0292-4) contains supplementary material, which is available to authorized users.
Highlights
MicroRNAs have been shown to play a critical role in the development and progression of nasopharyngeal carcinoma (NPC)
Expressed miRNAs between NPC and controls In order to isolate tumor cells from non-tumor cells, Laser capture microdissection (LCM) was performed on NPC and chronic nasopharyngitis specimens (Fig. 1a)
Validation of expression of miRNAs To confirm the deep sequencing results, we used qRT-PCR to assess the expression of 8 miRNAs with an independent cohort
Summary
MicroRNAs (miRNAs) have been shown to play a critical role in the development and progression of nasopharyngeal carcinoma (NPC). Accumulating studies have been performed on the molecular mechanisms of NPC, the miRNA regulatory networks in cancer progression remain largely unknown. Multiple genetic and epigenetic abnormalities synergistically disrupt normal cell function, especially playing an important role in NPC pathogenesis [3, 4]. MicroRNAs (miRNAs) have been increasingly recognized as important genetic regulators in the mammalian system [5]. Dysregulation of miRNA expression in NPC can regulate tumor cell growth, differentiation and apoptosis. Previous studies mostly focused on single miRNA, such as miR-138, a potential tumor suppressor by targeting CCND1 [7]. It is necessary to use high-tech tools to get a comprehensive miRNA analysis of NPC etiology
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